The mechanism of v-Ha-ras transformation of NHBE cells was investigated by testing the effect of v-Has-ras expression on chromosomal stability. To determine the effect of v-Ha-ras on chromosome structure plasmid H1 containing v-Ha-ras was transfected by protoplast fusion, and mitotic NHBE cells were examined 24 hours later to observe effects on chromosomal structure. Increased numbers of chromosome breaks and gaps were observed in v-Ha-ras transfected NHBE cells. Multistage progression of v-Ha-ras-transfected NHBE cells was studied by characterization of the tumorigenic growth in nude mice, cell surface antigens, and biochemical properties of TBE-1, TBE-1SA, and tumor cell lines derived from TBE-series cells. An in vitro model was developed to study the multistage progression in malignancy of human bronchial epithelial cells that were transformed to immortal cell lines with measurable malignant potential following transfection with Harvey ras oncogene (v-Ha- ras). Progressively malignant cell lines derived from this transformation were selected by continued growth in tissue culture (TBE-1), anchorage-independent growth in soft agar (TBE- 1SA), and xenogeneic transfer of TBE-series tumor tissues between mice. The TBE-1SA cell line has a shorter average latency period for subcutaneous primary tumors in athymic nude mice, higher frequency of successful transplantation, and more frequent metastasis to the liver, spleen, and lungs from primary tumors than tumorigenic cell lines selected for progression by continued growth in cell culture. The secondary growth of tumors that were passaged between mice also led to increased malignancy for each type of TBE-cell line tested.